Equalizing growth in high‐throughput small scale cultivations via precultures operated in fed‐batch mode

An often underestimated problem when working with different clones in microtiter plates and shake flask screenings is the non‐parallel and non‐equal growth of batch cultures. These growth differences are caused by variances of individual clones regarding initial biomass concentration, lag‐phase or specific growth rate. Problems arising from unequal growth kinetics are different induction points in expression studies or uneven cultivation periods at the time of harvest. Screening for the best producing clones of a library under comparable conditions is thus often impractical or even impossible. A new approach to circumvent the problem of unequal growth kinetics of main cultures is the application of fed‐batch mode in precultures in microtiter plates and shake flasks. Fed‐batch operation in precultures is realized through a slow‐release system for glucose. After differently growing cultures turn to glucose‐limited growth, they all consume the same amount of glucose due to the fixed feed profile of glucose provided by the slow‐release system. This leads to equalized growth. Inherent advantages of this method are that it is easy to use and requires no additional equipment like pumps. This new technique for growth equalization in high‐throughput cultivations is simulated and verified experimentally. The growth of distinctly inoculated precultures in microtiter plates and shake flasks could be equalized for different microorganisms such as Escherichia coli and Hansenula polymorpha. Biotechnol. Bioeng. 2009;103: 1095–1102. © 2009 Wiley Periodicals, Inc.     

Development of a selection system for the detection of L-ribose isomerase expressing mutants of <Emphasis Type="Italic">Escherichia coli</Emphasis>

L-Arabinose isomerase (E.C. 5.3.1.14) catalyzes the reversible isomerization between L-arabinose and L-ribulose and is highly selective towards L-arabinose. By using a directed evolution approach, enzyme variants with altered substrate specificity were created and screened in this research. More specifically, the screening was directed towards the identification of isomerase mutants with L-ribose isomerizing activity. Random mutagenesis was performed on the Escherichia coli L-arabinose isomerase gene (araA) by error-prone polymerase chain reaction to construct a mutant library. To enable screening of this library, a selection host was first constructed in which the mutant genes were transformed. In this selection host, the genes encoding for L-ribulokinase and L-ribulose-5-phosphate-4-epimerase were brought to constitutive expression and the gene encoding for the native L-arabinose isomerase was knocked out. L-Ribulokinase and L-ribulose-5-phosphate-4-epimerase are necessary to ensure the channeling of the formed product, L-ribulose, to the pentose phosphate pathway. Hence, the mutant clones could be screened on a minimal medium with L-ribose as the sole carbon source. Through the screening, two first-generation mutants were isolated, which expressed a small amount of L-ribose isomerase activity.     

Enzymatic synthesis of β‐glucosylglycerol using a continuous‐flow microreactor containing thermostable β‐glycoside hydrolase CelB immobilized on coated microchannel walls

β‐Glucosylglycerol (βGG) has potential applications as a moisturizing agent in cosmetic products. A stereochemically selective method of its synthesis is kinetically controlled enzymatic transglucosylation from a suitable donor substrate to glycerol as acceptor. Here, the thermostable β‐glycosidase CelB from Pyrococcus furiosus was used to develop a microstructured immobilized enzyme reactor for production of βGG under conditions of continuous flow at 70°C. Using CelB covalently attached onto coated microchannel walls to give an effective enzyme activity of 30 U per total reactor working volume of 25 µL, substrate conversion and formation of transglucosylation product was monitored in dependence of glucosyl donor (2‐nitrophenyl‐β‐D ‐glucoside (oNPGlc), 3.0 or 15 mM; cellobiose, 250 mM), the concentration of glycerol (0.25–1.0 M), and the average residence time (0.2–90 s). Glycerol caused a concentration‐dependent decrease in the conversion of the glucosyl donor via hydrolysis and strongly suppressed participation of the substrate in the reaction as glucosyl acceptor. The yields of βGG were ≥80% and ≈60% based on oNPGlc and cellobiose converted, respectively, and maintained up to near exhaustion of substrate (≥80%), giving about 120 mM (30 g/L) of βGG from the reaction of cellobiose and 1 M glycerol. The structure of the transglucosylation products, 1‐O‐β‐D ‐glucopyranosyl‐rac‐glycerol (79%) and 2‐O‐β‐D ‐glucopyranosyl‐sn‐glycerol (21%), was derived from NMR analysis of the product mixture of cellobiose conversion. The microstructured reactor showed conversion characteristics similar to those for a batchwise operated stirred reactor employing soluble CelB. The advantage of miniaturization to the microfluidic format lies in the fast characterization of full reaction time courses for a range of process conditions using only a minimum amount of enzyme. Biotechnol. Bioeng. 2009;103: 865–872. © 2009 Wiley Periodicals, Inc.     

Development of an Escherichia coli expression system and thermostability screening assay for libraries of mutant xylanase

A thermostability screening assay was developed using an Escherichia coli expression system to express Streptomyces lividans xylanase A (XlnA). The screening system was tested using mutants randomized at position 49 of the S. lividans XlnA gene, a position previously shown to confer thermostability with a I49P point mutation. The library was cloned into an E. coli expression vector and transformed into XL1-blue bacteria. The resulting clones were screened for increased thermostability with respect to wild-type XlnA. Using this assay, we isolated the I49P mutant previously shown to be thermostable, as well as novel I49A and I49C mutants. The I49A and I49C mutants were shown to have 2.8- to 8-fold increase in thermostability over that of wild-type XlnA. The results show that the screening assay can selectively enrich for clones with increased thermostability and is suitable for screening small- to medium-sized libraries of 5000–20,000 clones. Journal of Industrial Microbiology & Biotechnology (2000) 25, 310–314. Received 18 May 2000/ Accepted in revised form 19 September 2000     

Engineering of Phosphoserine Aminotransferase Increases the Conversion of l‐Homoserine to 4‐Hydroxy‐2‐ketobutyrate in a Glycerol‐Independent Pathway of 1,3‐Propanediol Production from Glucose

Phosphoserine aminotransferase (SerC) from Escherichia coli (E. coli) MG1655 is engineered to catalyze the deamination of homoserine to 4‐hydroxy‐2‐ketobutyrate, a key reaction in producing 1,3‐propanediol (1,3‐PDO) from glucose in a novel glycerol‐independent metabolic pathway. To this end, a computation‐based rational approach is used to change the substrate specificity of SerC from l ‐phosphoserine to l ‐homoserine. In this approach, molecular dynamics simulations and virtual screening are combined to predict mutation sites. The enzyme activity of the best mutant, SerC_R42W/R77W, is successfully improved by 4.2‐fold in comparison to the wild type when l ‐homoserine is used as the substrate, while its activity toward the natural substrate l ‐phosphoserine is completely deactivated. To validate the effects of the mutant on 1,3‐PDO production, the “homoserine to 1,3‐PDO” pathway is constructed in E. coli by coexpression of SerC_R42W/R77W with pyruvate decarboxylase and alcohol dehydrogenase. The resulting mutant strain achieves the production of 3.03 g L^?1 1,3‐PDO in fed‐batch fermentation, which is 13‐fold higher than the wild‐type strain and represents an important step forward to realize the promise of the glycerol‐independent synthetic pathway for 1,3‐PDO production from glucose.     

A TLC bioautographic method for the detection of α‐ and β‐glucosidase inhibitors in plant extracts

Introduction – Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of β‐glucosidase inhibitors but not for α‐glucosidase. Nonetheless, α‐glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti‐viral infections. Objective – To develop a TLC bioautographic method to detect α‐ and β‐glucosidase inhibitors in plant extracts. Methodology – The enzymes α‐ and β‐d ‐glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of α‐d ‐glucosidase, and 37°C for 20 min in the case of β‐d ‐glucosidase. For detection of the active enzyme, solutions of 2‐naphthyl‐α‐D‐glucopyranoside or 2‐naphthyl‐β‐D‐glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for α‐d ‐glucosidase) or 1 : 4 (for β‐d ‐glucosidase) and sprayed onto the plate to give a purple background colouration after 2–5 min. Results – Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited α‐d ‐glucosidase and β‐d ‐glucosidase down to 0.1 µg. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 µg for α‐glucosidase and 50 µg for β‐glucosidase. Conclusion – The screening test was able to detect inhibition of α‐ and β‐glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.     

Sensitive fluorimetric assays for α‐glucosidase activity and inhibitor screening based on β‐cyclodextrin‐coated quantum dots

A fluorescence method was established for a α‐glucosidase activity assay and inhibitor screening based on β‐cyclodextrin‐coated quantum dots. p‐Nitrophenol, the hydrolysis product of the α‐glucosidase reaction, could quench the fluorescence of β‐cyclodextrin‐coated quantum dots via an electron transfer process, leading to fluorescence turn‐off, whereas the fluorescence of the system turned on in the presence of α‐glucosidase inhibitors. Taking advantage of the excellent properties of quantum dots, this method provided a very simple, rapid and sensitive screening method for α‐glucosidase inhibitors. Two α‐glucosidase inhibitors, 2,4,6‐tribromophenol and acarbose, were used to evaluate the feasibility of this screening model, and IC₅₀ values of 24 μM and 0.55 mM were obtained respectively, which were lower than those previously reported. The method may have potential application in screening α‐glucosidase inhibitors. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of a major glucose transporter in Flavobacterium johnsoniae: Inhibition of F. johnsoniae colony spreading by glucose uptake

Many members of the phylum Bacteroidetes, such as Flavobacterium johnsoniae, can glide over a solid surface: an ability called gliding motility. It can be usually observed on agar plates as thin, flat, spreading colonies with irregular, feathery edges; this phenomenon is called colony spreading. Colony spreading of F. johnsoniae on 1.5% agar plates containing poor nutrients is dose‐dependently inhibited by addition of D‐glucose, as previously reported. Accordingly, here, we created mutants (by transposon mutagenesis) that partially suppressed glucose‐mediated inhibition of colony spreading. Among the isolates, we found that one had a transposon insertion in Fjoh_4565, tentatively named mfsA, which encodes a major facilitator superfamily (MFS) transporter previously shown to be required for growth on glucose, N‐acetyl‐glucosamine, and chitin. We constructed an mfsA deletion mutant and found that the mutant showed no glucose‐mediated acceleration of growth or glucose uptake. The mfsA gene complemented the phenotype of a glucose‐negative Escherichia coli. These results suggest that the mfsA gene encodes the sole MFS transporter of glucose in F. johnsoniae and that glucose uptake is partially required for the glucose‐mediated inhibition of F. johnsoniae colony spreading.

     

PURIFICATION AND CHARACTERIZATION OF β‐GLUCOSIDASE FROM GREATER WAX MOTH Galleria mellonella L. (LEPIDOPTERA: PYRALIDAE)

The greater wax moth, Galleria mellonella, is one of the most ruinous pests of honeycomb in the world. Beta‐glucosidases are a type of digestive enzymes that hydrolytically catalyzes the beta‐glycosidic linkage of glycosides. Characterization of the beta‐glucosidase in G. mellonella could be a significant stage for a better comprehending of its role and establishing a safe and effective control procedure primarily against G. mellonella and also some other insect pests. Laboratory reared final instar stage larvae were randomly selected and homogenized for beta‐glucosidase activity assay and subsequent analysis. The enzyme was purified to apparent homogeneity by salting out with ammonium sulfate and using sepharose‐4B‐l ‐tyrosine‐1‐naphthylamine hydrophobic interaction chromatography. The purification was 58‐fold with an overall enzyme yield of 29%. The molecular mass of the protein was estimated as ca. 42 kDa. The purified beta‐glucosidase was effectively active on para/ortho‐nitrophenyl‐beta‐d ‐glucopyranosides (p‐/o‐NPG) with Km values of 0.37 and 1.9 mM and Vmax values of 625 and 189 U/mg, respectively. It also exhibits different levels of activity against para‐nitrophenyl‐β‐d ‐fucopyranoside (p‐NPF), para/ortho‐nitrophenyl β‐d ‐galactopyranosides (p‐/o‐NPGal) and p‐nitrophenyl 1‐thio‐β‐d ‐glucopyranoside. The enzyme was competitively inhibited by beta‐gluconolactone and also was very tolerant to glucose against p‐NPG as substrate. The Ki and IC₅₀ values of δ‐gluconolactone were determined as 0.021 and 0.08 mM while the enzyme was more tolerant to glucose inhibition with IC50 value of 213.13 mM for p‐NPG.     

Cellobiose metabolism in Erwinia: genetic study

Summary The study of mutants of Erwinia specifically unable to ferment cellobiose indicates that the mutations are clustered between arg and ile on the chromosome of this organism. In vivo cloning of the genes responsible for cellobiose utilization lead to a plasmid, pBEC2, which complements all Erwinia Clb^- specific mutants. When introduced into wild-type E. coli it allows this organism to use cellobiose, arbutin and salicin; it also complements bglB and bglC mutants of Escherichia coli indicating that arbutin and salicin utilization is due to the products of the pBEC2 cloned genes. From the characterization of mutants pleiotropically affected in the utilization of various carbon sources, including cellobiose, arbutin and salicin, it is proposed that the three--glucosides are substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS).     

D-Amino acid dehydrogenase of Escherichia coli K12: positive selection of mutants defective in enzyme activity and localization of the structural gene

Summary A method for the positive selection of dadA mutants defective in Dolor-amino acid dehydrogenase has been devised. It consists in isolating mutants resistant to -chroro-Dolor-alanine and screening for mutant colony color on a special agar medium. All 70 Escherichia coli K12 dadA mutants isolated either by this method or by other selection procedures map at a locus which is near to hemA and closely linked with dadR. Since some of the dadA mutants are thermosensitive in Dolor-methionine utilization in vivo and have thermolabile Dolor-amino acid dehydrogenase in vitro, it is proposed that the dadA gene codes for the enzyme structure. The broad substrate specificity, apparent membrane localization, inducibility by alanine, and repressibility by glucose strongly suggest that the Dolor-amino acid dehydrogenase coded by the dadA gene is a species variant of the enzyme described under the same name in Salmonella typhimurium. It may be identical or homologous with the enzymes described under the names alaninase, Dolor-alanine oxidase or Dolor-alanine dehydrogenase in E. coli K12 or B.     

Extending the substrate scope of a Baeyer–Villiger monooxygenase by multiple-site mutagenesis

Baeyer–Villiger monooxygenase-catalysed reactions are attractive for industrial processes. Here we report on expanding the substrate scope of phenylacetone monooxygenase (PAMO). In order to introduce activity on alicyclic ketones in PAMO, we generated and screened a library of 1,500 mutants. Based on recently published structures of PAMO and its mutants, we selected previously uncharacterised positions as well as known hot-spots to be targeted by focused mutagenesis. We were able to mutate 11 positions in a single step by using the OmniChange method for the mutant library generation. Screening of the library using a phosphate-based activity detection method allowed identification of a quadruple mutant (P253F/G254A/R258M/L443F) active on cyclopentanone. The substrate scope of this mutant is extended to several aliphatic ketones while activity on aromatic compounds typical for PAMO was preserved. Moreover, the mutant is as thermostable as PAMO. Our results demonstrate the power of screening structure-inspired, focused mutant libraries for creating Baeyer–Villiger monooxygenases with new specificities.     

Chemostat selection of Escherichia coli mutants secreting thymidine,cytosine, uracil,guanine, and thymine

Escherichia coli mutants which secreted thymidine, thymine, uracil, cytosine, and guanine into the culture medium were isolated. The isolation strategy was based on the combination of a sensitive screening method and a mutant-generating system. The screening method made use of a thyA mutant of E. coli. These cells, when spread on the agar surface with the 3-galactosidase indicator X-gal, will grow into bule colonies if a minute amount of thymidine is supplied to them from a nearby secretor colony. A chemostat was used as a mutant-generating system to select for E. coli mutants that were resistant to inhibitors of the pyrimidine biosynthetic pathway. Although many mutants were selected based on their secretion of thymidine, other kinds of nucleosides and nucleobases, such as cytosine, uracil, guanine, and thymine, were also present in larger quantities. This rational selection strategy should be applicable to other species of micro-organisms for the isolation of better producers of nucleosides. The production of nucleosides and nucleobases by fermentation could then become a possibility.     

A double‐mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions

Mitogen‐activated protein kinase cascades are conserved in all eukaryotes. In Arabidopsis thaliana there are approximately 80 genes encoding MAP kinase kinase kinases (MAP3K), 10 genes encoding MAP kinase kinases (MAP2K), and 20 genes encoding MAP kinases (MAPK). Reverse genetic analysis has failed to reveal abnormal phenotypes for a majority of these genes. One strategy for uncovering gene function when single‐mutant lines do not produce an informative phenotype is to perform a systematic genetic interaction screen whereby double‐mutants are created from a large library of single‐mutant lines. Here we describe a new collection of 275 double‐mutant lines derived from a library of single‐mutants targeting genes related to MAP kinase signaling. To facilitate this study, we developed a high‐throughput double‐mutant generating pipeline using a system for growing Arabidopsis seedlings in 96‐well plates. A quantitative root growth assay was used to screen for evidence of genetic interactions in this double‐mutant collection. Our screen revealed four genetic interactions, all of which caused synthetic enhancement of the root growth defects observed in a MAP kinase 4 (MPK4) single‐mutant line. Seeds for this double‐mutant collection are publicly available through the Arabidopsis Biological Resource Center. Scientists interested in diverse biological processes can now screen this double‐mutant collection under a wide range of growth conditions in order to search for additional genetic interactions that may provide new insights into MAP kinase signaling.     

An integrated high throughput strategy to screen mutants of Paenibacillus polymyxa with high polymyxin E-productivity

An integrated high-throughput screening (HTS) strategy was developed to screen large numbers of polymyxin E-producing mutants of Paenibacillus polymyxa. Various types of mutants were transferred onto the surfaces of solidified agar in 96-well microtiter plates, and then inoculated to 96-deep-well microtiter plates for micro-cultivation. The culture conditions were optimized for the production of polymyxin E. The supernatants from the micro-culture plates were transferred to 96-well bioassay microtiter plates containing Escherichia coli JM109 for high-throughput bioassay. By using this high-throughput screening (HTS) procedure, one best producer P. polymyxa PE 5.808 was identified from a large NTG mutated library with about 5,000 isolates. The volumetric productivity of polymyxin E of P. polymyxa PE 5.808 was 1,200 μg/ml in shake flasks, about 140% improvement compared with that of the wild type strain.     

New and highly efficient methodology for screening high-yield strains of cytotoxic deacetylmycoepoxydiene (DAM)

Aims: To establish a highly efficient methodology for screening high yield strains of cytotoxic deacetylmycoepoxydiene (DAM), to meet the need of research on its mechanism of anti‐tumor properties and in vivo toxicity studies. Methods and Results: A simple, sensitive, and highly repetitive screening procedure ‘Antimicrobial‐TLC–HPLC’ (ATH) was established for the rapid obtaining of high‐yielding DAM mutants to replace the time and labor intensive anti‐tumor activity assay (MTT). With this ATH method, four highly yielding DAM mutants were selected out of 5000 total mutants, one of which, M4‐143, showed yields of more than 300 times (250·3 mg l^?1) that of the parent strain A123. Conclusions: The ATH method developed in this work has proven to be both economical and highly efficient with the screening of 1200 mutants in a one week time period, thusly shortening the expenditure of time and labor, without missing a single high‐yield mutant. Due to these characteristics, it is superior to other HTS screening methods described in earlier literature. The mutant M4‐143 has a good genetic stability and can be used for further research. Significance and Impact of the Study: This ATH screening method is not only perfect for screening high‐yield DAM mutants, but also, it is suitable to screen the strain libraries for those strains that have the ability to produce natural metabolites with antitumor activity.     

C‐terminal tail derived from the neighboring subunit is critical for the activity of Thermoplasma acidophilumD‐aldohexose dehydrogenase

The D ‐aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is a homotetrameric enzyme that catalyzes the oxidation of several D ‐aldohexoses, especially D ‐mannose. AldT comprises a unique C‐terminal tail motif (residues 247–255) that shuts the active‐site pocket of the neighboring subunit. The functional role of the C‐terminal tail of AldT has been investigated using mutational and crystallographic analyses. A total of four C‐terminal deletion mutants (Δ254, Δ253, Δ252, and Δ249) and two site‐specific mutants (Y86G and P254G) were expressed by Escherichia coli and purified. Enzymatic characterization of these mutants revealed that the C‐terminal tail is a requisite and that the interaction between Tyr86 and Pro254 is critical for enzyme activity. The crystal structure of the Δ249 mutant was also determined. The structure showed that the active‐site loops undergo a significant conformational change, which leads to the structural deformation of the substrate‐binding pocket. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Improved catalytic efficiency and active site modification of 1,4-beta-D-glucan glucohydrolase A from Thermotoga neapolitana by directed evolution

Thermotoga neapolitana 1,4-beta-d-glucan glucohydrolase A preferentially hydrolyzes cello-oligomers, such as cellotetraose, releasing single glucose moieties from the reducing end of the cello-oligosaccharide chain. Using directed evolution techniques of error-prone PCR and mutant library screening, a variant glucan glucohydrolase has been isolated that hydrolyzes the disaccharide, cellobiose, at a 31% greater rate than its wild type (WT) predecessor. The mutant library, expressed in Escherichia coli, was screened at 85 degrees C for increased hydrolysis of cellobiose, a native substrate rather than a chromogenic analog, using a continuous, thermostable coupled enzyme assay. The V(max) for the mutant was 108 +/- 3 units mg(-1), whereas that of the WT was 75 +/- 2 units mg(-1). The K(m) for both proteins was nearly the same. The k(cat) for the new enzyme increased by 31% and its catalytic efficiency (k(cat)/K(m)) for cellobiose also rose by 31% as compared with the parent. The nucleotide sequence of two positive clones and two null clones identified 11 single base shifts. The nucleotide transition in the most active clone caused an isoleucine to threonine amino acid substitution at position 170. Structural models for I170T and WT proteins were derived by sequence homology with Protein Data Bank code 1BGA from Paenibacillus polymyxa. Analysis of the WT and I170T model structures indicated that the substitution in the mutant enzyme repositioned the conserved catalytic residue Asn-163 and reconfigured entry to the active site.